PROJECT SUMMARY Islet-reactive T cells contribute to the development and progression of T1D. These cells can be found at low frequencies in the peripheral blood and potentially provide a window into the progression of disease and beta cell destruction. We propose in this application to characterize islet antigen reactive memory CD4+ (IARM- CD4) T cells in individuals with T1D using single cell RNA sequencing (RNAseq). This powerful tool will allow us to determine whether unique transcript signatures define IARM-CD4 T cells and if these signatures change over time with disease progression. Several preliminary findings indicate that an expanded analysis of these cells will provide unique information on the pathogenic processes in T1D: these cells in T1D donors have expanded TCR clonotypes not seen in healthy control donors, they have unique transcript phenotypes, and they exhibit imbalanced allelic expression of immune modulators, including T1D risk alleles. We propose three independent, but interrelated specific aims (SA): Determine the frequency, stability and specificity of expanded TCR clonotypes in IARM-CD4 T cells. We will isolate rare IARM-CD4 T cells from the peripheral blood of T1D and healthy control (HC) subjects and evaluate the stability of clonotype expansion over time and the relationship of clonotype expansion to the extent and/or rate of C-peptide loss. Characterize the transcript phenotypes of IARM-CD4 T cells and determine the generality, stability and clonotype specificity of these signatures. We will characterize the transcript signatures for IARM-CD4 T cells from individual subjects over time to determine their robustness, association with expanded TCR clonotypes, as well as the extent and/or rate of C-peptide loss. Determine the extent and stability of imbalanced allelic expression of immune genes, including T1D risk alleles, in IARM-CD4 T cells. We will characterize allelic imbalance in immune gene expression, including informative T1D risk alleles. Together, we anticipate these studies will help elucidate unique characteristics of IARM-T cells, provide insight into the immunological aspects of T1D progression, and to determine the feasibility of using IARM-CD4 T cells as biomarkers and therapeutic targets.